Alpha-methylacyl Coenzyme A Racemase Research Articles

Prognostic value of Alpha-Methyl acyl-Coenzyme A Racemase (AMACR) versus P53 in colorectal neoplasia

Prognostic value of Alpha-Methyl acyl-Coenzyme A Racemase (AMACR) versus P53 in colorectal neoplasia

Accurate prostate cancer detection based on enrichment and characterization of prostate cancer specific circulating tumor cells.

The low specificity of serum PSA resulting in the inability to effectively differentiate prostate cancer from benign prostate conditions is a persistent clinical challenge. The low sensitivity of serum PSA results in false negatives and can miss high-grade prostate cancers. We describe a non-invasive test for detection of prostate cancer based on functional enrichment of prostate adenocarcinoma associated circulating tumor cells (PrAD-CTCs) from blood samples followed by their identification by immunostaining for pan-cytokeratins (PanCK), prostate specific membrane antigen (PSMA), alpha methyl-acyl coenzyme-A racemase (AMACR), epithelial cell adhesion molecule (EpCAM), and common leucocyte antigen (CD45). Analytical validation studies were performed to establish the performance characteristics of the test using VCaP prostate cancer cells spiked into healthy donor blood (HDB). The clinical performance characteristics of the test were evaluated in a case-control study with 160 known prostate cancer cases and 800 healthy males, followed by a prospective clinical study of 210 suspected cases of prostate cancer. Analytical validation established analyte stability as well as acceptable performance characteristics. The test showed 100% specificity and 100% sensitivity to differentiate prostate cancer cases from healthy individuals in the case control study and 91.2% sensitivity and 100% specificity to differentiate prostate cancers from benign prostate conditions in the prospective clinical study. The test accurately detects PrAD-CTCs with high sensitivity and specificity irrespective of stage, serum PSA or Gleason score, which translates into low risks of false negatives or overdiagnosis. The high accuracy of the test could offer advantages over PSA based prostate cancer detection.

Tissue clearing techniques for three-dimensional optical imaging of intact human prostate and correlations with multi-parametric MRI.

Tissue clearing technologies have enabled remarkable advancements for in situ characterization of tissues and exploration of the three-dimensional (3D) relationships between cells, however, these studies have predominantly been performed in non-human tissues and correlative assessment with clinical imaging has yet to be explored. We sought to evaluate the feasibility of tissue clearing technologies for3Dimaging of intact human prostate and the mapping of structurally and molecularly preserved pathology data with multi-parametric volumetric MR imaging (mpMRI). Whole-mount prostates were processed with either hydrogel-based CLARITY or solvent-based iDISCO. The samples were stained with a nuclear dye or fluorescently labeled with antibodies against androgen receptor, alpha-methylacyl coenzyme-A racemase, or p63, and then imaged with 3D confocal microscopy. The apparent diffusion coefficientand Ktrans maps were computed from preoperative mpMRI. Quantitative analysis of cleared normal and tumor prostate tissue volumes displayed differences in 3D tissue architecture, marker-specific cell staining, and cell densities that were significantly correlated with mpMRI measurements in this initial, pilot cohort. 3D imaging of human prostate volumes following tissue clearing is a feasible technique for quantitative radiology-pathology correlation analysis with mpMRI and provides an opportunity to explore functional relationships between cellular structures and cross-sectional clinical imaging.

Diagnostic utility of immunochemical technique using p63 and Alpha Methylacyl Coenzyme A Racemase (AMACR) in the diagnosis of core-needle biopsy of the prostate: Experience in a tertiary academic institution in Nigeria

Background: Prostate adenocarcinoma is one of the most common malignancies and a leading cause of cancer-related mortality among males worldwide. There are challenges associated with confident/equivocal diagnosis of prostate carcinoma on small prostate samples from core-needle biopsies diagnosed histologically because of certain mimickers of prostatic carcinoma. Hence, there is a need to improve the diagnostic accuracy of the histological diagnosis of core-needle biopsies by utilizing immunohistochemical profiling to overcome these challenges. The aim of this study was to use p63 and alpha-methylacyl coenzyme A racemase (AMACR) immunostains to confirm hematoxylin and eosin (H and E) diagnosed adenocarcinomas and clarify equivocal diagnoses as well as correlate the H and E diagnoses with immunohistochemical diagnoses. Materials and Methods: This was a 3-year retrospective study of core-needle prostatic biopsies processed at the Anatomic and Molecular Pathology Department of Lagos University Teaching Hospital, Lagos, Nigeria. The formalin-fixed paraffin-embedded tissue blocks were retrieved, and new slides were prepared in cases where old slides were faded. The routinely processed slides were reviewed and classified into the following categories: benign, malignant (adenocarcinoma), and equivocal lesions (i.e., lesions considered suspicious for adenocarcinoma). The cases diagnosed as adenocarcinoma and equivocal lesions were then subjected to immunohistochemistry (IHC) using p63 and AMACR monoclonal antibodies to confirm the diagnoses of prostate adenocarcinoma and clarify the equivocal diagnoses. Based on the findings on IHC, the cases were reclassified as either adenocarcinoma, benign or indeterminate lesions (i.e., lesions that could not be classified as either benign or adenocarcinoma due to poor staining quality). Results: A total of 221 prostatic core biopsies met the inclusion criteria for this study. Out of these, histological diagnoses of prostatic adenocarcinoma were made in 113 cases (51.1%), 86 cases (38.9%) were benign, while equivocal cases accounted for 22 cases (10%). The result showed that out of 113 H and E diagnosed prostatic carcinoma that were subjected to p63 and AMACR stains, 101 (89.4%) of them were found to be truly adenocarcinoma, while 7 (6.2%) were benign and 5 cases (4.4%) were indeterminate lesions. The results of p63 and AMACR on the 22 histologically diagnosed equivocal prostatic lesions showed that 13 (59.1%) of the cases were adenocarcinoma, 7 cases (31.8%) were benign while 2 cases (9.1%) were indeterminate lesions. These p63 and AMACR immunostain results on routinely diagnosed prostatic carcinoma and equivocal diagnoses showed a statistically significant difference in the diagnostic potential of p63 and AMACR IHC when compared to the H and E as a diagnostic tool (P ≤ 0.001). Conclusion: We conclude that although histopathological examination of H and E sections remains the gold standard in the diagnosis of prostatic adenocarcinoma, the adjunctive use of p63 and AMACR immunostains is of great value in confirming small foci of adenocarcinoma, resolving morphologically equivocal cases and excluding benign mimickers as confounder in the diagnosis prostatic adenocarcinoma in small prostate samples obtained by core-needle biopsy.

Differentiation of renal cell tumors with morphological cocktails using a minimal panel of immunohistochemical markers.

Context:Morphological cocktails in renal cell carcinoma (RCC).Aims:Minimal immunohistochemistry (IHC) panel to resolve the diagnosis of renal cell cacinoma (RCC) with morphological overlaps.Settings and Design:RCC is the most common malignancy in kidney accounting for 90% of all kidney cancers. Clear cell RCC is the most common histological type followed by papillary RCC. However, many of the RCCs show morphological cocktails which may pose diagnostic difficulties in small biopsies and even in the resection specimens. Accurate diagnosis has both prognostic and therapeutic implications; hence, correct differentiation is necessary.Subjects and Methods:This retrospective study includes all renal cell tumors diagnosed on core biopsies, radical and partial nephrectomies between January 2015 and September 2017 were studied. The demographic, clinical, and gross findings were noted. The cases that had morphological overlap among the subtypes were subjected to a panel of IHC markers, including CD10, CK7, alpha-methyl acyl-coenzymeA racemase (AMACR), and CD117.Results:There were 128 RCC in the study period, and morphological overlap was seen in 36 (27.9%) specimens including 13 core biopsies, 16 radical, and 7 partial nephrectomies. IHC resolved 35/36 (97.2%) cases rendering a diagnosis of clear cell (11), papillary (15), chromophobe (4), and oncocytoma (5). However, in one case where the provisional diagnosis was oncocytic tumor, all IHC markers were negative rendering IHC noncontributory.Conclusions:Difficulty in diagnosis was encountered in many core biopsies, resection specimens which when subjected to IHC panel of CD10, CK7, AMACR, and CD117 helped in resolving the diagnosis of subtypes of RCC.

Current status and new approaches in prostate cancer diagnosis

Abstract Prostate adenocarcinoma, remaining among top most common cancers, is a heterogeneous group of tumors with a diverse morphological structure. Basing on the histological architecture of cancer tissue, individual cases can be classified into different therapeutic groups. Current diagnosis of prostate cancer brings many challenges. The major problem is the lack of effective and accessible diagnostic methods that would eliminate incidences of overdiagnosis and prevent unnecessary treatments of many patients. There are many efforts to determine favorable and unfavorable molecular prognostic factors. The basic marker currently used in this field is prostate-specific antigen (PSA). Increased level of PSA may suggest the presence of prostate cancer although its level is not specific for the disease and can be elevated also in certain benign hyperplastic or inflammatory conditions as well as after irritation or rectal examination. Clinical symptoms such as dysuria or hematuria are often uncharacteristic and benign prostatic diseases which cannot be confirmed on the basis of physical examination alone. Also, we often deal with the situation of false negative results of prostate needle biopsy, which require many tests to determine the final correct diagnosis. Moreover, prostate cancer can also be present in patients with non-elevated serum PSA level. Due to such difficulties, the search for new molecular markers that could be used for diagnostic purposes is underway. Evaluation of survivin level in prostate cancer tissue may serve as a new diagnostic indicator of prostate cancer progression. Other useful molecular biomarkers with good potential in prostate cancer diagnosis are AMACR (Alpha Methyl Acyl Coenzyme A Racemase), p-63 or Ki-67 or microRNAs present in body fluids.

Abstract 492: Chronic inflammation contributes to neoplastic progression in prostate tissue: a needle biopsy study

Abstract The relationship between inflammation and prostate cancer has not been established, although chronic inflammation has frequently been identified in prostate biopsies, radical prostatectomy and benign prostatic hyperplasia specimens. In the peripheral zone of the prostate, sometimes adjacent to foci of high-grade PIN and cancer, certain morphologic changes are often identified, which may represent active and terminal phases of chronic inflammation. These changes are designated as proliferative inflammatory atrophy (PIA) and post atrophic hyperplasia (PAH), and their morphology is well documented. In our previous studies, we have identified chronic inflammation as a putative contributor to neoplastic progression in prostate epithelial cells, and hypothesized that its adverse effects were related to an increase in Bcl2, a survival protein involved in cell survival and carcinogenesis. We hypothesize that changes in the stromal microenvironment, characterized by infiltration of immune cells, with generation of reactive oxygen species, can induce oxidative stress in the surrounding proliferating epithelium and cause permanent genomic alterations. In the present study, we focused on several key proteins involved in the inflammatory process, COX2 and iNOS; cell survival, Bcl2 and GSTPπ; and evaluated immunohistochemical expression of alpha-methylacyl coenzyme A racemase (AMACR) and basal cell-specific markers 34βE12 and/or p63 to evaluate possible neoplastic alterations in epithelial cells in an inflammatory environment. We evaluated 16 prostate core needle biopsy specimens that exhibited the presence of chronic inflammation as well as PIA and PAH lesions along with immunohistochemical staining for various markers was performed in each set of biopsies. The integrity of the basal layer was maintained in the area of chronic inflammation with high expression of p63 in 72% of these cells. Approximately 68% of luminal cells expressed high to moderate levels of iNOS and COX-2, whereas 55% of these cells express modest levels of GSTP1 and Bcl2. Interestingly, prostatic glands near the areas of chronic inflammation in the PIA lesions exhibit high AMACR expression in luminal cells and weak to patchy p63 expression in basal cells, which was associated with increased expression of the inflammatory markers COX2 and iNOS, as well as loss in pro-survival signal GSTP1 and Bcl2 in the adjacent luminal cells. These neoplastic alterations were observed in 6/16 (38%) of the needle biopsy specimens. Our findings suggest that basal cells undergo alterations in a setting of chronic inflammation. This is important because basal cells are considered to be progenitor cells capable of differentiating into secretory luminal cells, but under the influence of chronic inflammation, they may instead transform into the neoplastic cells that characterize high grade prostatic intraepithelial neoplasia and prostatic adenocarcinoma. Citation Format: Shardul C. Soni, Michael Glover, Qinghu Ren, Gregory T. MacLennan, Pingfu Fu, Sanjay Gupta. Chronic inflammation contributes to neoplastic progression in prostate tissue: a needle biopsy study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 492. doi:10.1158/1538-7445.AM2017-492

Expression of Alpha - Methylacyl - Coenzyme A Racemase (AMACR) in Colorectal Neoplasia.

Red meat and dairy products have been implicated in colonic cancers. They are rich in branched chain fatty acids which require an enzyme Alpha-Methylacyl-Coenzyme A Racemase (AMACR) for their oxidation. Increased expression of AMACR in colorectal premalignant lesions and carcinomas suggests its possible role in carcinogenesis. To study the expression of AMACR in colorectal neoplasia and its correlation with the histological grade, stage and nodal status of colorectal malignancy. All cases of colorectal neoplasms were included in the study. AMACR expression was studied in 56 cases which included 44 cases of adenocarcinoma and 12 cases of adenoma and a normal colonic mucosal tissue was used as a control. A tissue microarray was prepared by manual method. AMACR expression was studied by Immunohistochemistry (IHC) and it was correlated with the grade, stage and nodal status of the cancer. Chi-square test was used for analysis or results. The p-value of <0.05 was considered significant. Out of 12 cases of adenoma 25% (3/12) were negative; 50% (6/12) showed poor expression, 25% (3/12) had moderate expression and none showed strong expression. Nearly, 34.1% (15/44) case of carcinoma were negative; 45.45% (20/44) had poor; 13.63% (6/44) had moderate and 6.81% (3/44) had a strong expression. Normal colonic tissue showed no expression. There was no significant difference of AMACR expression between adenoma and carcinoma cases. AMACR expression was found to be increased in low grade carcinomas (G1 and G2). G1 showed AMACR positivity in 62% cases and G2 showed 71.4% positivity. No association was found between AMACR expression and different American Joint Committee on Cancer (AJCC) stages and nodal status of CRC. Increased expression of AMACR in adenomas and carcinomas as compared to non-neoplastic epithelium of colon implies that, it plays a role in colorectal neoplasia. Decreased expression of AMACR in high grade carcinomas suggests its role in differentiation of the tumour.

Evaluation of role of alpha-methyl acyl-coenzyme A racemase/P504S and high molecular weight cytokeratin in diagnosing prostatic lesions.

In recent years basal cell markers (high molecular weight cytokeratin [HMWCK]) and prostate biomarker alpha-methyl acyl-coenzyme A racemase (AMACR) have been used as adjuvant to morphology in diagnostically challenging cases with a very high sensitivity and specificity. This has increased the diagnostic accuracy of prostate cancer worldwide. In this prospective study, total of 50 cases including 37 cases of malignant lesions and 13 cases of benign lesions of the prostate were taken. Tumor grade was determined according to Gleason's grading system. AMACR and HMWCK expressions were determined by immunohistochemical staining. The obtained results were analyzed and evaluated using Chi-square statistical test (SPSS version 20). AMACR was not expressed in any of the 13 cases of benign lesions of the prostate while in malignant lesions of prostate it was expressed in 33 of 37 (89.18%) cases. All 4 (100%) cases of well-differentiated carcinoma were positive for AMACR expression. 21 of 25 (84%) moderately differentiated and all 10 (100%) cases of poorly differentiated tumors were positive for AMACR. There was statistically significant difference in expression of AMACR between benign and malignant lesions of the prostate, indicated byP = 0.001. In benign lesions, HMWCK was expressed in all the 13 (100%) cases while in malignant lesions of prostate it was not expressed in any of the (0%) case. All 13 benign lesions were positive for HMWCK only. AMACR expression was not seen in any of the benign lesion. Out of 37 malignant cases, 4 cases were negative for both, 33 cases were positive only for AMACR, but no case was positive only for HMWCK. As an adjunct to biopsy, AMACR and HMWCK have value for resolving diagnostically challenging cases.

Gene expression profiling of clear cell papillary renal cell carcinoma: comparison with clear cell renal cell carcinoma and papillary renal cell carcinoma

Clear cell papillary renal cell carcinoma is a distinct variant of renal cell carcinoma that shares some overlapping histological and immunohistochemical features of clear cell renal cell carcinoma and papillary renal cell carcinoma. Although the clear cell papillary renal cell carcinoma immunohistochemical profile is well described, clear cell papillary renal cell carcinoma mRNA expression has not been well characterized. We investigated the clear cell papillary renal cell carcinoma gene expression profile using previously identified candidate genes. We selected 17 clear cell papillary renal cell carcinoma, 15 clear cell renal cell carcinoma, and 13 papillary renal cell carcinoma cases for molecular analysis following histological review. cDNA from formalin-fixed paraffin-embedded tissue was prepared. Quantitative real-time PCR targeting alpha-methylacyl coenzyme-A racemase (AMACR), BMP and activin membrane-bound inhibitor homolog (BAMBI), carbonic anhydrase IX (CA9), ceruloplasmin (CP), nicotinamide N-methyltransferase (NNMT), schwannomin-interacting protein 1 (SCHIP1), solute carrier family 34 (sodium phosphate) member 2 (SLC34A2), and vimentin (VIM) was performed. Gene expression data were normalized relative to 28S ribosomal RNA. Clear cell papillary renal cell carcinoma expressed all eight genes at variable levels. Compared with papillary renal cell carcinoma, clear cell papillary renal cell carcinoma expressed more CA9, CP, NNMT, and VIM, less AMACR, BAMBI, and SLC34A2, and similar levels of SCHIP1. Compared with clear cell renal cell carcinoma, clear cell papillary renal cell carcinoma expressed slightly less NNMT, but similar levels of the other seven genes. Although clear cell papillary renal cell carcinoma exhibits a unique molecular signature, it expresses several genes at comparable levels to clear cell renal cell carcinoma relative to papillary renal cell carcinoma. Understanding the molecular pathogenesis of clear cell papillary renal cell carcinoma will have a key role in future sub-classifications of this unique tumor.

Alpha-methylacyl coenzyme A racemase overexpression in gallbladder carcinoma confers an independent prognostic indicator

AimsIncreased β-oxidation of branched-chain fatty acids provides an additional metabolic advantage for cancer cells thereby enhancing tumour development and progression. Alpha-methylacyl coenzyme A racemase (AMACR) is an enzyme essential for...

Clear cell adenocarcinoma of the bladder and urethra: cases diffusely mimicking nephrogenic adenoma

Although clear cell adenocarcinoma have been described focally mimicking nephrogenic adenoma, we have identified a subset of clear cell adenocarcinoma that diffusely resembles nephrogenic adenoma (nephrogenic adenoma-like clear cell adenocarcinoma). Twelve classic clear cell adenocarcinomas of the bladder and urethra and 7 nephrogenic adenoma-like clear cell adenocarcinomas were compared to 10 nephrogenic adenomas. Classic clear cell adenocarcinomas and nephrogenic adenoma-like clear cell adenocarcinomas comprised 4 men and 15 women. The following features were seen in classic clear cell adenocarcinomas: nephrogenic adenoma-like clear cell adenocarcinomas: predominantly solid pattern (7/12:0/7), marked nuclear pleomorphism (7/12:1/7), prominent nucleoli (5/12:1/7), clear cytoplasm in 50% or greater of tumor (7/12:0/7), and necrosis (8/12:3/7), although the necrosis in nephrogenic adenoma-like clear cell adenocarcinomas was often focal and intraluminal. Both patterns of clear cell adenocarcinomas showed prominent hobnail features, although more pronounced in nephrogenic adenoma-like clear cell adenocarcinomas. Muscularis propria invasion was seen in 5 of 9 classic clear cell adenocarcinomas and 6 of 6 nephrogenic adenoma-like clear cell adenocarcinomas, where evaluable. Classic clear cell adenocarcinoma was associated with urothelial carcinoma (n = 2) and endometriosis (n = 1). The Ki-67 rate in clear cell adenocarcinomas ranged from 10% to 80% compared with 0% to 5% in nephrogenic adenoma. The following antibodies were not helpful in distinguishing nephrogenic adenoma-like clear cell adenocarcinoma from nephrogenic adenoma: CD10, estrogen receptor, p63, high-molecular-weight cytokeratin, and alpha-methylacyl coenzyme-A racemase. PAX2 expression was more frequent in nephrogenic adenoma (89%) compared to both patterns of clear cell adenocarcinoma (29%-32%). The key features discriminating between nephrogenic adenoma-like clear cell adenocarcinoma and nephrogenic adenoma include occasional clear cells, more prominent pleomorphism especially hyperchromatic enlarged nuclei, and extensive muscular invasion. Presence of mitoses and a high rate of Ki-67 expression in lesions resembling nephrogenic adenoma require clinical correlation, close follow-up, and repeat biopsy with more extensive sampling.

Immunohistochemical Antibody Cocktail Staining (p63/HMWCK/AMACR) of Ductal Adenocarcinoma and Gleason Pattern 4 Cribriform and Noncribriform Acinar Adenocarcinomas of the Prostate

Overexpression of alpha-methylacyl coenzyme A racemase (AMACR) in combination with absence of basal cell markers [ie, p63 and high molecular weight cytokeratin (HMWCK)] is typical of classic acinar prostatic adenocarcinoma. We studied the expression and diagnostic utility of p63/HMWCK/AMACR immunohistochemical cocktail staining in ductal adenocarcinoma and cribriform Gleason pattern 4 acinar prostate cancer and compared it to noncribriform Gleason pattern 4 acinar prostate cancer. One to 4 representative formalin-fixed paraffin-embedded archival tissue blocks from 62 radical prostatectomy specimens harboring prostate cancer of ductal (n=51), cribriform Gleason pattern 4 acinar (n=27), and noncribriform Gleason pattern 4 acinar adenocarcinoma (n=48) were included in this study. Immunohistochemistry was performed using a triple stain of AMACR, p63, and HMWCK. Only staining that was moderate or strong was considered positive. The percentage of staining intensity and the presence of occasional basal cells positive with p63/HMWCK were recorded in each histologic type of prostatic adenocarcinoma. Seventy-seven percent of ductal prostatic adenocarcinoma, 67% of cribriform acinar prostatic carcinoma, and 81% of noncribriform acinar prostatic carcinoma showed positive staining for AMACR. There was no statistically significant difference between AMACR staining among the 3 histologic types, although there was a trend for noncribriform acinar prostatic carcinoma to have greater expression of AMACR than cribriform acinar prostatic carcinoma (P=0.07). Staining was often heterogeneous, varying in staining intensities within the same histologic type of carcinoma. Basal cells were detectable by p63 and HMWCK in a patchy fashion in 31.4% (16/51) of ductal and 29.6% (8/27) of cribriform acinar carcinomas compared with 2.1% (1/48) of noncribriform acinar carcinomas. In summary: (1) the majority of prostatic ductal and cribriform acinar carcinomas strongly expressed AMACR, however, subpopulations of these prostatic carcinoma were either completely negative or only weakly positive; (2) AMACR staining was often heterogeneous in intensity in the same histologic type of tumor, even within the same case; (3) patchy basal cell staining in noncribriform acinar prostatic carcinoma is rare. In contrast, remnants of basal cells identified by p63/HMWCK were seen in a patchy fashion in a significant minority of both ductal and cribriform acinar prostatic adenocarcinoma, which most likely represents intraductal spread of tumor.

Optimization of laser capture microdissection and RNA amplification for gene expression profiling of prostate cancer

BackgroundTo discover prostate cancer biomarkers, we profiled gene expression in benign and malignant cells laser capture microdissected (LCM) from prostate tissues and metastatic prostatic adenocarcinomas. Here we present methods developed, optimized, and validated to obtain high quality gene expression data.ResultsRNase inhibitor was included in solutions used to stain frozen tissue sections for LCM, which improved RNA quality significantly. Quantitative PCR assays, requiring minimal amounts of LCM RNA, were developed to determine RNA quality and concentration. SuperScript II™ reverse transcriptase was replaced with SuperScript III™, and SpeedVac concentration was eliminated to optimize linear amplification. The GeneChip® IVT labeling kit was used rather than the Enzo BioArray™ HighYield™ RNA transcript labeling kit since side-by-side comparisons indicated high-end signal saturation with the latter. We obtained 72 μg of labeled complementary RNA on average after linear amplification of about 2 ng of total RNA.ConclusionUnsupervised clustering placed 5/5 normal and 2/2 benign prostatic hyperplasia cases in one group, 5/7 Gleason pattern 3 cases in another group, and the remaining 2/7 pattern 3 cases in a third group with 8/8 Gleason pattern 5 cases and 3/3 metastatic prostatic adenocarcinomas. Differential expression of alpha-methylacyl coenzyme A racemase (AMACR) and hepsin was confirmed using quantitative PCR.

Is Triple Immunostaining With 34βE12, p63, and Racemase in Prostate Cancer Advantageous?

This study aimed to determine the usefulness of a combination of 3 immunohistochemical markers, 34/betaE12, p63 and alpha-methylacyl coenzyme A racemase (AMACR), for the diagnosis of prostate cancer using tissue microarrays (TMAs) constructed from 91 archival radical prostatectomy specimens derived from the Pathology Department files of Singapore General Hospital, Singapore. Triple immunostaining using a cocktail of these 3 antibodies was performed on TMA sections using the streptavidin-biotin method. When compared with immunohistochemical staining using the individual antibodies, we found that the triple cocktail allowed improved evaluation of basal cells in benign glands, and AMACR allowed simultaneous corroboration of malignant prostatic glands in the same section. We achieved a specificity of 100% with the triple cocktail, and sensitivity was acceptable, at 93.8%. In comparison, specificity and sensitivity of the individual antibodies were 95.5% and 97.3%, 93.3% and 93.8%, 97.0% and 95.6% for p63, 34betaE12, and AMACR, respectively. The triple cocktail offers a cost-effective way of evaluating abnormal prostatic glandular foci, in addition to maximizing the use of small tissue samples from prostatic needle biopsies.

Atypical small acinar proliferation: biopsy artefact or distinct pathological entity?

To determine if atypical small acinar proliferation (ASAP) represents minimally sampled prostate cancer not fully evaluated on a biopsy or a distinct pathological entity, by examining prostates removed at radical cystectomy, as a finding of ASAP of the prostate on needle-core biopsy is closely associated with the detection of cancer on subsequent biopsy. In all, 65 consecutive cystoprostatectomy specimens taken from June 1990 to March 2004 had prostatic material reviewed by one genitourinary pathologist (S.E.M.). The presence of high-grade prostatic intraepithelial neoplasia (HGPIN), ASAP, and adenocarcinoma was recorded. Foci of ASAP found in the absence of cancer were assessed with additional sectioning, high-molecular weight keratin (CK903), and alpha-methylacyl coenzyme A racemase (AMACR) immunohistochemistry. In all, 24 of 65 specimens (37%) had adenocarcinoma. Of the 41 without cancer, 18 (44%) had neither HGPIN nor ASAP, 14 (34%) had HGPIN alone, three (7%) had ASAP alone (four foci), and six had both HGPIN and ASAP (15%). Two foci of ASAP were not present on any further sectioning. The remaining eight foci all lacked CK903 stain, indicating disruption of the basal cell layer. Of these eight, only five were present for the AMACR stain, all of which were positive. Two of these five developed into a lesion considered cancer on further sectioning. ASAP identified in incidental prostates represented marginally sampled cancer in at least two of 10 foci assessed. The remainder could not be resolved as benign on further evaluation, and remained suspicious for malignancy.

Differential Expression of α-Methylacyl Coenzyme A Racemase in Adenocarcinomas of the Small and Large Intestines

Alpha-methylacyl coenzyme A racemase (AMACR), a novel immunomarker for prostatic adenocarcinoma, has recently been shown to be expressed in a number of malignancies including colorectal adenocarcinoma. In the current study, 59 surgically resected primary small intestinal adenocarcinomas (34 ampullary and 25 non-ampullary) were immunohistochemically examined for AMACR expression and compared with 66 colorectal adenocarcinomas (including 24 secondary tumors involving the small intestine by direct extension or metastasis). The results show that no AMACR immunoreactivity was detected in normal-appearing small and large intestinal mucosa. While 41 of 66 (62%) colorectal adenocarcinomas exhibited a variable degree of cytoplasmic staining, only 1 of 25 (4%) non-ampullary and 2 of 34 (6%) ampullary small intestinal adenocarcinomas showed positive AMACR immunoreactivity (P < 0.0001). Interestingly, AMACR appeared to be less frequently expressed in mucinous or poorly differentiated colorectal adenocarcinomas when compared with non-mucinous or better-differentiated counterparts, suggesting an association with microsatellite instability status. These results extend our previous observations that small intestinal adenocarcinomas differ markedly from colorectal adenocarcinomas despite their morphologic similarity. The different AMACR expression patterns may not only provide an additional diagnostic tool in the distinction between adenocarcinomas of the small and large intestinal origins but may also shed light on further understanding of intestinal tumorigenesis.

PROSTATE CANCER DETECTION ON URINALYSIS FOR α METHYLACYL COENZYME A RACEMASE PROTEIN

We assessed the feasibility of a novel urinary test for prostate cancer based on the presence of alpha methylacyl coenzyme A racemase (AMACR) protein in voided urine specimens obtained after prostate biopsy. Clean catch voided urine specimens were prospectively collected from 26 consecutive men immediately after transrectal ultrasound guided prostate biopsy for suspected malignancy. The presence of AMACR was evaluated in a blinded manner by Western blot analysis and correlated with biopsy results and patient clinical information. AMACR was detected in the urine in 18 of 26 patients (69%). AMACR was detected in all 12 patients with biopsy confirmed adenocarcinoma of the prostate (100% sensitivity, 95% CI 75 to 100), in 5 of 12 with no evidence of cancer on biopsy (58% specificity, 95% CI 29 to 78) and in 1 of 2 (50%, 95% CI 3 to 80) with atypia on biopsy. Overall AMACR detection was associated with cancer status by prostate biopsy in 21 of 26 patients (86%). We report the feasibility of a novel, noninvasive, nonprostate specific antigen based molecular approach to detect prostate cancer in voided urine. To our knowledge this is the first report of AMACR protein detection in the urine of patients with prostate cancer. A screening test based on urinary AMACR may develop into a useful adjunct to serum prostate specific antigen and digital rectal examination for identifying men at increased risk for harboring prostate cancer despite negative biopsy. Such a test has potential application for stratifying patients into low and high risk groups for surveillance vs repeat biopsy.

Alpha-Methylacyl coenzyme A racemase as a tissue biomarker for prostate cancer.

Molecular profiling of prostate cancer has led to the identification of candidate biomarkers and regulatory genes. Discoveries from these genome-scale approaches may have applicability in the analysis of diagnostic prostate specimens. To determine the expression and clinical utility of alpha-methylacyl coenzyme A racemase (AMACR), a gene identified as being overexpressed in prostate cancer by global profiling strategies. Four gene expression data sets from independent DNA microarray analyses were examined to identify genes expressed in prostate cancer (n = 128 specimens). A lead candidate gene, AMACR, was validated at the transcript level by reverse transcriptase polymerase chain reaction (RT-PCR) and at the protein level by immunoblot and immunohistochemical analysis. AMACR levels were examined using prostate cancer tissue microarrays in 342 samples representing different stages of prostate cancer progression. Protein expression was characterized as negative (score = 1), weak (2), moderate (3), or strong (4). Clinical utility of AMACR was evaluated using 94 prostate needle biopsy specimens. Messenger RNA transcript and protein levels of AMACR; sensitivity and specificity of AMACR as a tissue biomarker for prostate cancer in needle biopsy specimens. Three of 4 independent DNA microarray analyses (n = 128 specimens) revealed significant overexpression of AMACR in prostate cancer (P<.001). AMACR up-regulation in prostate cancer was confirmed by both RT-PCR and immunoblot analysis. Immunohistochemical analysis demonstrated an increased expression of AMACR in malignant prostate epithelia relative to benign epithelia. Tissue microarrays to assess AMACR expression in specimens consisting of benign prostate (n = 108 samples), atrophic prostate (n = 26), prostatic intraepithelial neoplasia (n = 75), localized prostate cancer (n = 116), and metastatic prostate cancer (n = 17) demonstrated mean AMACR protein staining intensity of 1.31 (95% confidence interval, 1.23-1.40), 2.33 (95% CI, 2.13-2.52), 2.67 (95% CI, 2.52-2.81), 3.20 (95% CI, 3.10-3.28), and 2.50 (95% CI, 2.20-2.80), respectively (P<.001). Pairwise comparisons demonstrated significant differences in staining intensity between clinically localized prostate cancer compared with benign prostate tissue, with mean expression scores of 3.2 and 1.3, respectively (mean difference, 1.9; 95% CI, 1.7-2.1; P<.001). Using moderate or strong staining intensity as positive (score = 3 or 4), evaluation of AMACR protein expression in 94 prostate needle biopsy specimens demonstrated 97% sensitivity and 100% specificity for detecting prostate cancer. AMACR was shown to be overexpressed in prostate cancer using independent experimental methods and prostate cancer specimens. AMACR may be useful in the interpretation of prostate needle biopsy specimens that are diagnostically challenging.